Project Summary Objectives Tuberculosis (TB) remains a leading cause of infectious mortality worldwide. At present, the only available vaccine for TB, Bacillus Calmette-Guerin (BCG), has not proven effective in the prevention of adult tuberculosis. Increasingly, military personnel have been deployed to areas of the world where TB is endemic, with recent examples including Iraq and Afghanistan. As a result, an improved vaccine for TB is urgently needed. We have recently identified and characterized human, M. tuberculosis (Mtb)-reactive, non-classically restricted Cluster of differentiation 8+ (CD8+) T cells restricted by the non-classical Human Leukocyte Antigen 1b (HLA-Ib) molecule MR1. This molecule has not previously been shown to present pathogen associated antigens. In this application, we will define the role of MHC class I-related protein 1 (MR1) in the recognition of Mtb infected cells, and will define the antigen presented by MR1. AIM 1: Define the role of MR1 in CD8+ T cell recognition of Mtb-infected cells. a) Determine whether or not MR1-restricted Mtb-reactive T cells are innately acquired. b) Define the relative contribution of MR1, HLA-E, and CD1 to HLA-Ib-restricted CD8+ T cell TB immunity. c) Determine if primary lung epithelial cells can present Mtb antigens in the context of MR1, and if MR1- restricted CD8+ T cells are present in the lung. AIM 2: Define the antigen processing pathway for MR1. a) Identify the sub-cellular location in which TB antigens and MR1 become associated. b) Define the role of the proteasome, do-novo protein synthesis, TAP, and endosomal acidification in the processing of MR1 antigen(s). AIM 3: Determine the Mtb-derived antigen(s) that is (are) presented by MR1. a) Use an M. smegmatis transposon mutagenesis library to identify the MR1 antigen (s). Approach MR1-resctricted T cells from human cord blood, from peripheral blood adults with evidence of infection with Mtb, and in peripheral blood from those without infection will be compared by T-cell Receptor Excision Circles (TREC) for evidence of prior replication. Blocking antibodies will be used to determine the relative contribution of each HLA-Ib allele. Tracheal epithelial cells, type II pneumoncytes, and alveolar macrophages will be prepared, and tested for their ability to process and present Mtb and antigens found within the Mtb cell wall. Flow cytometry will be used to enumerate CD8 T cells expressing the MR-1 associated V17 T-cell Receptor (TCR), and these cells tested for their ability to recognize Mtb infected cells using IFN-3 ELISPOT. A combination of confocal microscopy and flow organellometry will be used to evaluate the presence or absence of MR1 in the Mtb phagosome. Fluorescently tagged MR1 suitable for lentivirial expression will be developed. Using chemical blockers as well as protein blockers of TAP, the requirement for TAP, acidificaton, and de-novo protein synthesis will be determined. A genetic approach will be used to define the mycobacterial MR1 antigen. In collaboration with Dr David Sherman (Seattle Biomedical Research Institute) an M. smegmatis transposon library will be developed, and tested for loss of function against MR1 restricted T cell clones. Confirmation of these results will be performed with Dr Karen Dobos (Colorado State University) and Bill Bishai (Johns Hopkins) using available mutants from a mariner transposon library.